Journal of Natural Proa’uds Vol. 58, No. 1,pp. 124-127, January I995
124
ISOLATION AND STRUCTURE IDENTIFICATION OF TWO NEW DERIVATIVES OF THE MYCOTOXIN FUSAROCHROMENONE PRODUCED BY FUSARIUM EQUISETI WEIPING &E, CHESTER J. hllROCH.4,
Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota 55 108 and YECHUN WEN Shanghai Research Centre of Biotechnology, Chinese Academy of Sciences, Shanghai, 200233, Peoplei Republic of China ABSTRACT.-TWO new chromone derivatives, 2,2-dimethyl-5-amino-6-(4’hydroxylbutycyl)-4-chromone E21 and 2,2dimethyl-5-amino-6-( 2’E-ene-4’-hydroxylbutyryl)4-chromone [4], were isolated from rice cultures of Fusarium equiseti. Their structures were deduced from chemical and spectral data.
Fusarochromanone { 17is a mycotoxin produced by an isolate of Fusarium equiseti (Corda)Sacc. Alaska 2-2 (originally identified as F. graminearum) (1-5). This toxin causes tibial dyschondroplasia, a common bone disease in chicks, and reduced hatchability offertile chicken eggs under experimental conditions (2). Fusarochromanone [l]has also been detected in chicken feed samples associatedwith tibial dyschondroplasia (1).It is a 4-chromone derivative with some unusual substituents. Several fusarochromanone derivatives have been found in cultures of F. equiseti (3,GS). Two minor metabolites, 2 and 4 , were also isolated as yellow powders from a rice culture of the fungal isolate. Compound 2 is light yellow and exhibited a blue fluorescence under uv light. Compound 4 is bright yellow but did not fluoresce under uv. Eims of 2 resulted in the observation ofa [MI’ at mlz 277 and [M-H,O}+ ion at mlz 259 indicating the mol wt to be 277 daltons with the presence of a hydroxy group. This conclusion was supported by fabms which exhibited a [M+H}+ ion at m l z 278 and an [M+H-H,O]+ ion at mlz 260; and by pcims which yielded a [MfH)’ ion at mlz 278, a {M+C,H$: ion at mlz 306 and a [M+H-H,O] ion at mlz 260; as well as ncims which yielded a {MI- ion at mlz 277. Hreims of 2 gave the [MI’ at mlz
1 R,=NH,, R,=H 2
R,=R,=H
3 R,=H,R,=Ac
4 R=H 5 R=Ac
277.1309 (calculated as 277.1314 for C15H1&04, with seven double bond equivalents). Acetylation of 2 yielded a monoacetate 137 with a {MI’ ion at mlz 319 by eims, also supporting the mol wt of 2 as 277 daltons and the presence of a hydroxy substituent. The very similar eims spectra of 2 and 1 (2) suggested that their structures were closely related. The fragment ion at mlz 218, which resulted from an a-cleavage between C- 1’ and C2’, were base peaks in the eims spectra of both 1 (2) and 2 suggesting that 2 had the same 4-chromone basic structure and similar substitutents to 1. A fragment ion [M-CH,CHOH]+ at mlz 233.1017
January 19951
Xie et a/.: Chromones from Fusarium
(calculated as 233.105 1 for Cl3H1,NO3) in the hreims of 2 was suggestive of a 4’hydroxylbutyryl unit in the molecule. The ‘H-nmr spectrum of 2 showed signals forC-2[(CH3)J, C-3 (CH,), C-7 (H), C-8 (H), and C-5 (NH,), which agreed with the ‘H-nmr spectrum of 1 (2) and confirmed that they had the same chromone skeleton. Proton resonances at 1.95-2.18ppm(2H,m), 2.99ppm(2H, t,J=7.0Hz)and3.71 ppm(2H,tJ=6.0 Hz) confirmed the presence of a 6-(4’hydroxylbutyryl) unit in 2. The. ‘H-nmr spectrum of 3 showed a singlet at 2.04 pprn (3H), confirming the acetylation and presence of one -OH group in 2. A downfield shift of the singlet from 3.7 1 ppm (C-4’ protons) in the ‘H-nmr spectrum of 2 to 4.14 pprn (C-4’ protons) in the ‘H-nmr spectrum of 3 confirmed the O H group at C-4. Therefore, the structure of 2 was established as 2,2-dimethyl-5 -amino-6-(4’-hydroxylbutyryl)4-chromone. The eims of 4 showed a [MIi ion at mlz 27 5 and a [M- H201+ion at mlz 257, indicating the mol wt of 4 to be 275 daltons, again with the presence of an -OH group. Supporting evidence came from the fabms which exhibited a [MSH]’ ion a t mlz 276 and a [M+H-H,Of+ ionatmlz258aswellas the ncims which showed aIM1- ion at mlz 275. Acetylation of 4 yielded a monoacetate 151, identified by eims, and which confirmed the presence of a hydroxy substituent in 4. Most of the fragment ions, including that at rnlz 218 in the eims spectrum of 4 , were also found for 2, indicating that they were analogous except for a structural difference in the 6-butyryl group. The eims of 4 had a [MI’ ion at mlz 275 as the base peak, suggesting a more stable structure compared with 2. This increased stability was consistent with increased unsaturation in the butyryl group. The eims of 4 gave an intense fragment ion at mlz 188 (40) resulting from cleavage between C-6 and C-1’ and a weaker ion at mlz 218 (30),
125
that may be postulated as being due to cleavage between C-1’ and C-2’. This fragmentation pattern differed from that of 2, where rnlz 218 was the base peak while rnlz 188 was very weak. This difference in their ms fragmentation patterns agreed with the proposed structures of 2 and 4. In the eims of 1 and 2, cleavage between C-6 and C-1 ’ was reduced due to conjugation between the carbonyl at C1’ and the ring, while cleavage between C-1’ and C-2’ was very much favored. In the eims of 4 , cleavage between C- 1’ and C-2’ was reduced due to conjugation between the carbonyl at C-1’ and the C2’, C-3’ double bond, while cleavage between C-6 and C-1 ’ increased because of reduced competition. Accurate mass measurement of 4 by hreims, which gave the [M}’ at rnlz 275.1086 (calculated as 275.1015 for C1,Hl,NO4,with 8 double bond equivalents), supported the presence of one more double bond in 4 compared with 2. Comparison of the ‘H- and 13 C-nmr spectra of 4 with published data of 1 (2) showed good agreement and supported the 4-chromone skeleton of 4. The ‘H-nmr spectrum of 4 showed signals at 4.43-4.45 ppm (2H, m), 6.97 pprn ( l H , dt, J 4 5 . 2 , 3.7, and 3.7 Hz) and 7.17 pprn ( l H , d,J=15.2 Hz), and supported a partial structure of -COCH=CHCH,OH. The C-2’, C-3’ double bond was in the E form since J2f3f=15.2Hz was observed. In the ‘Hnmr spectrum of 5 , asignal at 4.79-4.81 ppm (H,-4’, m), which showed a downfield shift compared with the analogous signal in the ‘H-nmr spectrum of 4 , indicated that the -OH substituent was at C-4’. The 13C-nmr and DEPT nmr spectra of 4 showed signals at 104.01 pprn (s) and 124.59 pprn (s), which indicated the presence of a C-2’, C-3’ double bond. The uv spectrum of 4 , which exhibited absorbances at 260 and 390 nm in comparison with values 245 and 278 nm in the spectrum of 2, was also indicative of increased conjugation in 4. The structural relationship of 2 and 4 was
126
Journal of Natural Products
proved by generation of 2 through the hydrogenation of 4. The hydrogenation product was identified by eims. Therefore, the structure of 4 was established as 2,2-dimethyl-5 -amino-6-( 2'E-ene-4'hydroxylbutyryl)-4-chromone.
Wol. 58, No. 1
( l H , dt,J=15.2, 3.7, and 3.7 Hz, H-3'), 7.17 ( l H , d,]=15.2 Hz, H-2'), 7.93 ( l H , d,J=9.0 Hz, H-7),9.42(1H, brs,ArNHa),9.73 ( l H , brs, ArNHb); I3C n m r (CDCI,, 50 MHz) 6 26.58 (q), 49.02(t),65.28(t), 104.01 (d), 124.59(d), 140.46 (d), 144.14 (d), 79.41 (s), 104.56 (s), 112.16 (s), 155.46 (s), 166.04 (s), 189.76 (s) and 193.82 (s).
ACETYLATION OF COMPOUND 2.-Compound 2 (5 mg) was acetylated with (Ac),O (1 ml) in pyridine (1 ml) at room temperature for 18 h. T h e GENERALEXPERIMENTALPROCEDURES.--~~~~~ fluorescent product [3]was isolated by Si gel prep. spectra were measured on a VG 7070EQ instrutlc in CHC1,-MeOH (97:3). Compound 3 exhibment. Eims spectra were obtained at 70 eV. Fabms ited: eims (70 eV) m/z [MI' 319 ( 5 4 , 259 ( l l ) , spectra were obtained in thioglycerol as matrix. 244 (20), 233 (30), 218 (loo), 204 (lo), 202 (6), Methane was used as reactant gas in cims. 'H-, 190 (8), 177 (8), 162 (59X 106 (13); 'H nmr "C-, and DEPT nmr spectra were measured on a (CDCI,, 200 MHz) 6 1.45 (6H, s, H,-1 1 and H,Bruker AC-200 instrument at 200 MHz, using 12), 1.96-2.16 (ZH, m, H2-3'), 2.04 (3H, s, 4'TMS as internal standard. Uv spectra were reOCOCH,), 2.69(2H,s, H,-3),2.92 (2H, t,J=7.3 corded on a Beckman DB-GT spectrophotometer Hz, H,-2'), 4.14 (ZH, t,J=6.4 Hz, H2-4'), 6.06 in MeOH. ( l H , d,J=9.0 Hz, H-8), 7.84 ( l H , d,J=9.0 Hz, FUNGAL hfmmIhL-The fungal isolate was H-7), 9.36 ( l H , br s ArNHa), 9.55 ( l H , br s, F. equiseti (Alaska 2-2). ArNHb). EXTRACTION AND IsounON.-Procedures Ac~nuno~oFCOMPOUND4.~ompound for culture growth, extraction, and column frac4 (4 mg) was acetylated with N-acetylimidazole (3 tionation have been described in a previous report mg) in CHCI, (1 ml) at room temperature for 18 (8).The fractions containing 2 and 4, as detected h. The reaction product [SI that absorbed shortby tlc, were purified by repeated Si gel prep. tlc, wavelength uv light, was purified by Si gel prep. with a fluorescence indicator, in CHC1,-MeOH tlc in CHC1,-MeOH (19:l). Compound 5 exhib( 19:1).The tlc spots and bands of 2 were visualized ited: eims (70 eV) mlz [MI' 317 (loo), 262 (12), by long-wavelength uv. The spots and bands of 4 258 (37), 257 (84), 244 (42), 242 (66), 228 (14), were visualized by short-wavelength uv. Com218 (51), 214 (25), 202 (37), 201 (18), 191 (15), pounds 2 and 4 had Rfvalues of 0.74 and 0.47, 188 (36), 174 (28), 162 (45), 144 (11), 136 (13), respectively,onSigel tlc inCHC13-MeOH(19:l). 135 (13,118 (12), 106 (17); 'H nmr (CDCI,, 200 Five mg of 2 and 20 mg of 4 were obtained as MHz) 6 1.46(6H, s, H3-11 and H3-12),2.14 (3H, yellow powders. 5, 4'-OCOCH3), 2.70 (2H, S, H,-3), 4.794.81 (ZH, m, H2-4'), 6.09 ( l H , d,J=9.0 Hz, H-8), 2,2-Di~thy(-S-amina-6-(4'-bydroxylbutyry()6.85 (1H, dt,J=15.3, 4.7, and 4.7 Hz, H-3'), 4 - c h m n e [2].-Ir u max (film) 3397, 3285, 7.07 ( l H , d,J=15.3 Hz, H-2'), 7.86 ( l H , d, 2934, 2872, 1740, 1655, 1597, 1566, 1458, J=9.O Hz, H-7), 9.45 ( l H , br s, ArNHa), 9.67 1373, 1315, 1559, 1107, 995, 895 cm-'; uv h ( l H , br s, ArNHb). max (MeOH) 276, 246, 213 nm; rns mlz [MI(C,,H,$JO,) 277 (56), 259 (19), 244 (19), 233 HYDROGENATION OF 4.-A solution of 4 (1 (67), 218 (loo), 204(16), 190(14), 176(17), 162 mg) in MeOH (10 ml) with a catalytic amount of (80), 106 (16); 'H nmr (CDCI,, 50 MHz) 6 1.45 Pd/C (5%) was stirred for 2 h under H,. The crude (6H, s, H 3 - l l and H,-12), 1.95-2.18 ( l H , m, Hproduct was chromatographed by prep. tlc (Si gel) 3'), 2.69 (ZH, S, H2-3),2.99 (1H, t,J=7.0 Hz, Hto give pure 2. Z'), 3.71 (2H, t,J=6.0 Hz, H,-4'), 6.06 ( l H , d, J=8.9 Hz, H-8), 7.87 ( l H , d,J=8.9 Hz, H-7), ACKNOWLEDGMENTS 9.37 ( l H , br s, ArNHa), 9.58 ( l H , br s, ArNHb). Published as paper No. 21,184ofthecontri2,2-Dimetbyl-S-arnino-6-(2'E-ene-4'bution series of the Minnesota Agricultural Exhydroxy/buryryl)-4-cbrmone [4l.-Ir Y max (film) periment Station based on research conducted 3669, 3389, 3277, 2957, 2892, 1660, 1651, under Project 22-34H, supported by HATCH 1599, 1566, 1556, 1466, 1373, 1275, 1174, funds. 1159,1107and895cm-';uvXmax(MeOH)390, 260, and 210 nm; ms mlz [MI' (C,,H,,NO,) 275 LITERATURE CITED (loo),260 (lo), 257 (23), 244 (49), 242 (28), 220 1. P. Krogh, D.H. Christensen, B. Hald, B. (19), 218 (30), 201 (13), 188 (40), 162 (17), 106 Harlou, C. h e n , E.J. Pedersen, and U. (17 ) ;'H nmr (CDCI,, 200MHz)S 1.45 (6H, s, H,Thrane, AppL Environ. Microbial., 55, 3184 11 andH3-12),2.69(2H,s,H,-3),4.434.45 (ZH, (1989). m, H,-4'), 6.07 ( l H , d,J=9.0 Hz, H-8), 6.97
EXPERIMENTAL
January 19953 2.
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Xie et al. : Chromones from Fusarium
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